LOCUS       sequence-209429-        7255 bp    DNA     circular SYN 14-APR-2025
DEFINITION  FRT integration of wild type beta-globin reporter; enables detection
            of 5'-3' decay intermediates (xrFrag) and allows detection via 
            northern blot (4H binding sites).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7255)
  AUTHORS   Boehm V, Gerbracht JV, Marx MC, Gehring NH
  TITLE     Interrogating the degradation pathways of unstable mRNAs with 
            XRN1-resistant sequences.
  JOURNAL   Nat Commun. 2016 Dec 5;7:13691. doi: 10.1038/ncomms13691.
  PUBMED    27917860
REFERENCE   2  (bases 1 to 7255)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2016-12-5"; volume: "7:13691. doi"; pages: " 
            10.1038/ncomms13691"
COMMENT     Sequence Label: pcDNA5/FRT/TO-globin_del5UTR_WT-xrRNA-4H
FEATURES             Location/Qualifiers
     source          1..7255
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        384..587
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     538..558
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    589..607
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    610..628
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     638..662
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     complement(2976..2993)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2982..3206
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3339..3358)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3490..3537
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3545..4567
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3545..4567
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4697..4818
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4734..4753)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4788..4807
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4867..4883)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4867..4883)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4880..4902)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4891..4907
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4915..4921,4922..4939,4940..4945))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4960..4981
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5098..5115)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5269..5857)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5349..5368)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6028..6819,6820..6888))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6651..6670
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6889..6993)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7068..7087)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
      421 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
      481 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
      541 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc cctatcagtg
      601 atagagatct ccctatcagt gatagagatc gtcgacgagc tcgtttagtg aaccgtcaga
      661 tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca
      721 gcctccggac tctagcgttt aaacttaagc ttgctagcac catggtgcac ctgactcctg
      781 aggagaagtc tgccgttact gccctgtggg gcaaggtgaa cgtggatgaa gttggtggtg
      841 aggccctggg caggttggta tcaaggttac aagacaggtt taaggagacc aatagaaact
      901 gggcatgtgg agacagagaa gactcttggg tttctgatag gcactgactc tctctgccta
      961 ttggtctatt ttcccaccct taggctgctg gtggtctacc cttggaccca gaggttcttt
     1021 gagtcctttg gggatctgtc cactcctgat gctgttatgg gcaaccctaa ggtgaaggct
     1081 catggcaaga aagtgctcgg tgcctttagt gatggcctgg ctcacctgga caacctcaag
     1141 ggcacctttg ccacactgag tgagctgcac tgtgacaagc tgcacgtgga tcctgagaac
     1201 ttcagggtga gtctatggga cccttgatgt tttctttccc cttcttttct atggttaagt
     1261 tcatgtcata ggaaggggat aagtaacagg gtacagttta gaatgggaaa cagacgaatg
     1321 attgcatcag tgtggaagtc tcaggatcgt tttagtttct tttatttgct gttcataaca
     1381 attgttttct tttgtttaat tcttgctttc tttttttttc ttctccgcaa tttttactat
     1441 tatacttaat gccttaacat tgtgtataac aaaaggaaat atctctgaga tacattaagt
     1501 aacttaaaaa aaaactttac acagtctgcc tagtacatta ctatttggaa tatatgtgtg
     1561 cttatttgca tattcataat ctccctactt tattttcttt tatttttaat tgatacataa
     1621 tcattataca tatttatggg ttaaagtgta atgttttaat atgtgtacac atattgacca
     1681 aatcagggta attttgcatt tgtaatttta aaaaatgctt tcttctttta atatactttt
     1741 ttgtttatct tatttctaat actttcccta atctctttct ttcagggcaa taatgataca
     1801 atgtatcatg cctctttgca ccattctaaa gaataacagt gataatttct gggttaaggc
     1861 aatagcaata tttctgcata taaatatttc tgcatataaa ttgtaactga tgtaagaggt
     1921 ttcatattgc taatagcagc tacaatccag ctaccattct gcttttattt tatggttggg
     1981 ataaggctgg attattctga gtccaagcta ggcccttttg ctaatcatgt tcatacctct
     2041 tatcttcctc ccacagctcc tgggcaacgt gctggtctgt gtgctggccc atcactttgg
     2101 caaagaattc accccaccag tgcaggctgc ctatcagaaa gtggtggctg gtgtggctaa
     2161 tgccctggcc cacaagtatc actaagctcg ctttcttgct gtccaatttc tctcgaggtg
     2221 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtggactt gatagtcagg
     2281 ccagccggtt aggctgccac cgaaggttgg tagacggtgc tgcctgcgac caaccccagg
     2341 aggactgggt taccaaagct gattctccac ggttggaaag cctcccagaa ccgtctcgga
     2401 agaggagtcc ctgccaacaa tggagatgaa gcccgtgtca gatcgcgaaa gcgccacttc
     2461 gccgaggagt gcaatctgtg aggccccagg aggactgggt aaacagtcga gcaccccacc
     2521 agtgcaggct gcctatcaga aagtggtggc tggtgtggct aatgccctgg cccacaagta
     2581 tcactaagct cgctttcttg ctgtccaatt tgtcgagcac cccaccagtg caggctgcct
     2641 atcagaaagt ggtggctggt gtggctaatg ccctggccca caagtatcac taagctcgct
     2701 ttcttgctgt ccaatttgtc gagcacccca ccagtgcagg ctgcctatca gaaagtggtg
     2761 gctggtgtgg ctaatgccct ggcccacaag tatcactaag ctcgctttct tgctgtccaa
     2821 tttgtcgagc accccaccag tgcaggctgc ctatcagaaa gtggtggctg gtgtggctaa
     2881 tgccctggcc cacaagtatc actaagctcg ctttcttgct gtccaatttg tcgacccggg
     2941 cggccgcaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca
     3001 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     3061 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     3121 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     3181 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     3241 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     3301 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     3361 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     3421 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     3481 acgtacctag aagttcctat tccgaagttc ctattctcta gaaagtatag gaacttcctt
     3541 ggccaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga
     3601 cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga
     3661 tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga
     3721 tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag tgcttgacat
     3781 tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt
     3841 gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg aggccatgga
     3901 tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg
     3961 aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta
     4021 tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga
     4081 gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg
     4141 ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc
     4201 gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc
     4261 ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc
     4321 gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga
     4381 cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg
     4441 agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg
     4501 ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa
     4561 ggaatagcac gtactacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     4621 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     4681 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     4741 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     4801 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     4861 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     4921 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     4981 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     5041 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     5101 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     5161 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     5221 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     5281 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     5341 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     5401 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     5461 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     5521 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     5581 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     5641 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5701 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     5761 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     5821 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5881 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     5941 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     6001 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     6061 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     6121 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     6181 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     6241 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     6301 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     6361 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     6421 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     6481 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6541 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     6601 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     6661 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6721 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     6781 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6841 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     6901 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     6961 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     7021 cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
     7081 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
     7141 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
     7201 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgc
//
